Biological Therapeutics Research Articles

Investigating the impact of synonymous gene recoding on a recombinantly expressed monoclonal antibody under different process parameters

AbstractMonoclonal antibodies (mAbs) are commonly used biologic therapeutics with a wide variety of clinical applications. During the development process, manufacturers consider different production parameters to improve protein yield and achieve appropriate quality of the product. Synonymous gene recoding is one of such attributes that is often considered and implemented to enhance protein expression. However, it has to be used with caution, as it may lead to protein misfolding and ER stress, which complicates efforts to manufacture the desired mAb. To investigate how changing mRNA sequence composition under different protein production parameters might affect the quality of recombinantly produced mAbs, we performed a comprehensive and systematic study assessing impact of synonymous gene recoding (commonly referred to as codon optimization) strategies in the context of varied cell culture parameters on product quality, biochemical and functional characteristics. We report the impact of these parameters on mAb glycosylation profiles, charge variant profile, aggregation, fragmentation, and mAb functional response from combinations of different production parameters. These results uncovered a complex interplay of sequence composition and manufacturing parameters and emphasize the importance of assessing changes to key quality attributes when optimizing mAb manufacturing, including the use of synonymous gene recoding.

"Current and emerging drug therapies in Alzheimer's disease: A pathophysiological Perspective".

The analytical and experimental investigation of several targets and biomarkers that help in explaining significant cognitive deficits, covering drug development and precision medicine aimed at different chronic neurodegenerative conditions such as Alzheimer's disease (AD), Parkinson's disease, synaptic dysfunction, brain damage from neuronal apoptosis, and other disease pathologies; this served as the foundation for all phase studies. The focus of current therapeutic approaches is on developing humanized antibodies, agonist and antagonist drugs, receptors, signaling molecules, major targeted drug-metabolizing enzymes, and other metabolites to treat neurodegeneration in the AD brain brought on by tau hyperphosphorylation, amyloid plagues, or other cholinergic effects. The five A's-amnesia, agnosia, aphasia, apraxia, and anomia-are the typical symptoms associated with AD. While the main goal of drug therapeutics studies is modified amino acids acting as pro-drugs, pharmacokinetics studies and trends in evaluating drug-drug interactions focus on interactions between drugs and antibodies, drugs and therapeutic biologics like metabolites, herbs, interleukin-based, and gene silencing mechanism-based. Studies on the biotransformation of xenobiotic compounds and the metabolism of exogenous and endogenous substances are conducted under Phase I, Phase II, and Phase III trials because the pivotal pharmacokinetic properties of drugs, such as absorption, distribution, metabolism, and excretion (ADME), aid in understanding variations in the crucial improvement of various target drugs. This review also highlights the developments in soon-to-be genetically created targeted medications that may serve as ground-breaking treatments for cholinergic illnesses in the brains of AD patients and other neurodegenerative conditions.

Nuclear Magnetic Resonance Study of Monoclonal Antibodies Near an Oil-Water Interface.

Monoclonal antibodies (mAb) represent an important class of biologic therapeutics that can treat a variety of diseases including cancer, autoimmune disorders or respiratory conditions (e.g. COVID-19). However, throughout their development, mAb are exposed to air-water or oil-water interfaces that may trigger mAb partial unfolding that can lead to the formation of proteinaceous aggregates. Using a combination of dynamic surface tensiometry and spatially resolved 1D 1H NMR spectroscopy, this study investigates if adsorption of a model IgG2a-κ mAb to the oil-water interface affects its structure. Localized NMR spectroscopy was performed using voxels of 375 µm, incrementally approaching the oil-water interface. Dynamic interfacial tension progressively decreases at the oil-water interface over time, confirming mAb adsorption to the interface. Localized NMR spectroscopy results indicate that, while the number of mAb-related chemical resonances and chemical shift frequencies remain unaffected, spectral line broadening is observed as voxels incrementally approach the oil-water interface. Moreover, the spin-spin (T2) relaxation of the mAb molecule was measured for a voxel centered at the interface and shown to be affected differentially across the mAb resonances, indicating a rotational restriction for mAb molecules due to presence of the interface. Finally, the apparent diffusion coefficient of the mAb for the voxel centered at the interface is lower than the bulk mAb. These results suggest that this specific mAb interacts with and may be in exchange with bulk mAb phase in the vicinity of the interface. As such, these localized NMR techniques offer the potential to probe and quantify alterations of mAb properties near interfacial layers.

Utilization of Liquid Chromatography-Mass Spectrometry and High-Resolution Ion Mobility-Mass Spectrometry to Characterize Therapeutically Relevant Peptides with Asparagine Deamidation and Isoaspartate.

Rapid identification of asparagine (Asn) deamidation and isoaspartate (isoAsp) in proteins remains a challenging analytical task during the development of biological therapeutics. For this study, 46 therapeutically relevant peptides corresponding to 13 peptide families (13 unmodified peptides and 33 modified peptides) were obtained; modified peptides included Asn deamidation and isoAsp. The peptide families were characterized by three methods: reversed-phase ultrahigh performance liquid chromatography-mass spectrometry (RP-UHPLC-MS); flow injection analysis high-resolution ion mobility-mass spectrometry (FIA-HRIM-MS); and shortened gradient RP-UHPLC-HRIM-MS. UHPLC-MS data acquisition was 2 h per injection, in contrast to high-throughput 1 min data acquisition of the FIA-HRIM-MS technique. A rapid 2D peptide map has been demonstrated by combining shortened gradient RP-UHPLC with HRIM, to optimize the resolution of the Asn-, Asp-, and isoAsp-containing peptides, increasing the likelihood of detecting peptides containing these quality attributes with expedited data acquisition. Additionally, this paper provides an ion mobility calibration data set for therapeutically relevant peptides (unmodified and modified) over an ion-neutral collisional cross-section range of 300-800 Å2.

Integrating Micro- and Nanostructured Platforms and Biological Drugs to Enhance Biomaterial-Based Bone Regeneration Strategies.

Bone defects resulting from congenital anomalies and trauma pose significant clinical challenges for orthopedics surgeries, where bone tissue engineering (BTE) aims to address these challenges by repairing defects that fail to heal spontaneously. Despite numerous advances, BTE still faces several challenges, i.e., difficulties in detecting and tracking implanted cells, high costs, and regulatory approval hurdles. Biomaterials promise to revolutionize bone grafting procedures, heralding a new era of regenerative medicine and advancing patient outcomes worldwide. Specifically, novel bioactive biomaterials have been developed that promote cell adhesion, proliferation, and differentiation and have osteoconductive and osteoinductive characteristics, stimulating tissue regeneration and repair, particularly in complex skeletal defects caused by trauma, degeneration, and neoplasia. A wide array of biological therapeutics for bone regeneration have emerged, drawing from the diverse spectrum of gene therapy, immune cell interactions, and RNA molecules. This review will provide insights into the current state and potential of future strategies for bone regeneration.

Medicare Local Coverage Determinations: Evidence Quality Is Stronger For Covered Indications.

Under the 21st Century Cures Act of 2016, a summary of the evidence used to support local coverage determinations, which represent the vast majority of Medicare's coverage decisions for new technologies, must be made publicly accessible. Using reports from the Medicare Coverage Database on local coverage determinations and the medical literature, we examined the availability of these decisions and the quality of evidence cited for therapeutic drugs, biologics, and moderate- or high-risk devices during the period 2015-22 to understand whether evidence strength and generalizability differed for indications with favorable versus unfavorable coverage decisions. Evidence summaries were publicly available for 26percent of coverage decisions originally effected during 2015-18 and 100percent during 2019-22. Among the latter, when compared with noncovered indications, indications with favorable coverage decisions cited twice the number of evidence sources (median, four versus two) and more often cited studies with a mean patient age of sixty-five and older (78percent versus 47percent). Fewer than one-third of all studies reported race or ethnicity data. There was no significant association between indication coverage and the study design strength of cited clinical studies. Although the 21st Century Cures Act enhanced transparency, clinical evidence that better reflects the Medicare beneficiary population is needed to inform local coverage determinations.

Improving access to domestic innovative medicines: characteristics and trends of approved drugs in China 2010–2024

China has initiated drug regulatory reforms since 2015. Here, we analyze the characteristics and trends of domestic innovative drugs approved for marketing in China from January 2010 to May 2024 to explore the effectiveness of drug regulatory reform. Overall, 219 drugs were approved, with growth in chemicals and therapeutic biologics post-reform. Single-arm trials as an important option for clinical trial design of antineoplastic agents increased. The time for each link from investigational new drug (IND) to new drug application (NDA) has been shortened post-reform. Moreover, the time for access to medical insurance for approved drugs has been shortened and price reductions have been increased. China's drug regulatory reforms have made progress in improving the accessibility of domestic innovative drugs.

Advances in Development of Drug Treatment for Hemophilia with Inhibitors.

Patients with hemophilia A and B who have inhibitors face limited treatment options, because replacement therapy with clotting factor VIII or IX concentrates is ineffective, particularly for patients with high-titer inhibitors. Current mainstay therapies include immune tolerance induction (through frequent injections of clotting factor VIII or IX concentrates) to eradicate inhibitors and bypassing agents (such as recombinant activated clotting factor VII and activated prothrombin complex concentrates) for the prevention and treatment of bleeding episodes. The use of these agents typically requires intravenous injections and sometimes hospitalization, which can be burdensome for patients. More recently, emicizumab, a bispecific antibody that mimics the function of activated clotting factor VIII, has demonstrated favorable efficacy for prophylaxis in patients with hemophilia A and inhibitors, representing a promising new therapeutic strategy. Ongoing research aims to discover and develop easy-to-use nonfactor agents for managing hemophilia with inhibitors. This review summarizes the current understanding of the pathophysiology of inhibitor development in hemophilia, outlines existing treatment options, and discusses advancements in novel therapeutic biologics, including a recombinant activated clotting factor VII variant (marzeptacog alfa), a new bispecific antibody (Mim8), antitissue factor pathway inhibitor antibodies (concizumab and marstacimab), and small interfering RNA targeting antithrombin (fitusiran). All of these agents are administered subcutaneously, with some offering the convenience of less frequent dosing (e.g., weekly or monthly). These potential drug candidates may provide significant benefits for the prophylaxis or treatment of bleeding disorders in patients with hemophilia and inhibitors.

An Adhesive Hydrogel Technology for Enhanced Cartilage Repair: A Preliminary Proof of Concept.

Knee cartilage has limited natural healing capacity, complicating the development of effective treatment plans. Current non-cell-based therapies (e.g., microfracture) result in poor repair cartilage mechanical properties, low durability, and suboptimal tissue integration. Advanced treatments, such as autologous chondrocyte implantation, face challenges including cell leakage and inhomogeneous distribution. Successful cell therapy relies on prolonged retention of therapeutic biologicals at the implantation site, yet the optimal integration of implanted material into the surrounding healthy tissue remains an unmet need. This study evaluated the effectiveness of a newly developed photo-curable adhesive hydrogel for cartilage repair, focusing on adhesion properties, integration performance, and ability to support tissue regeneration. The proposed hydrogel design exhibited significant adhesion strength, outperforming commercial adhesives such as fibrin-based glues. An in vivo goat model was used to evaluate the hydrogels' adhesion properties and long-term integration into full-thickness cartilage defects over six months. Results showed that cell-free hydrogel-treated defects achieved superior integration with surrounding tissue and enhanced cartilage repair, with notable lateral integration. In vitro results further demonstrated high cell viability, robust matrix production, and successful cell encapsulation within the hydrogel matrix. These findings highlight the potential of adhesive hydrogel formulations to improve the efficacy of cell-based therapies, offering a potentially superior treatment for knee cartilage defects.

How Biologics Have Changed the Drug Discovery Landscape.

Advances in molecular biology and molecular genetics as well as major scientific breakthroughs in immunology and oncology have led to the rapid growth of biologic therapeutics. Their success has resulted in significant changes to virtually every step in the drug discovery and development process. Biologics are produced by living organisms, and screening libraries are generated by immunization or phage display. Lead optimization utilizes sophisticated protein engineering to improve drug-like properties and targeting specificity. The manufacturing process for biologics is complex and requires highly specialized facilities. Determination of pharmacology and safety must overcome the complications associated with species specificity. Initial clinical testing must proceed more slowly and carefully due to the limited predictive utility of preclinical data. In summary, the drug discovery and development process has been dramatically altered by biologic therapeutics and will continue to evolve with the introduction of messenger RNA-based therapeutics and the application of artificial intelligence.

Stellabody: A novel hexamer-promoting mutation for improved IgG potency.

Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation of IgG is a normal physiological aspect of IgG biology and recently described mutations that facilitate this process have a substantial impact upon monoclonal antibody behavior resulting in the elicitation of dramatically enhanced complement-dependent cytotoxicity, Fc receptor function, and enhanced antigen binding effects, such as targeted receptor agonism or microbe neutralization. Whereas the discovery of IgG hexamerisation enhancing mutations has largely focused on residues with exposure at the surface of the Fc-Fc and CH2-CH3 interfaces, our unique approach is the engineering of the mostly buried residue H429 in the CH3 domain. Selective substitution at position 429 forms the basis of Stellabody technology, where the choice of amino acid results in distinct hexamerisation outcomes. H429F results in monomeric IgG that hexamerises after target binding, so called "on-target" hexamerisation, while the H429Y mutant forms pH-sensitive hexamers in-solution prior to antigen binding. Moreover, Stellabody technologies are broadly applicable across the family of antibody-based biologic therapeutics, including conventional mAbs, bispecific mAbs, and Ig-like biologics such as Fc-fusions, with applications in diverse diseases.

Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

Material Trends and Clinical Costings in Systematically Identified CDER‐Approved Nanomedicines

AbstractResearch into mechanisms and potential applications of nanomaterials in medicine has expanded steadily in recent decades, with increasing translational focus. Development costs, including for clinical trials, are often cited as factors limiting the translational viability of novel nanomedicines, especially compared to small new molecular entities (NMEs) and therapeutic biologics. Yet, to date, there has been no systematic investigation into the clinical programs or costs of translating and commercializing nanomedicines, nor a comparison to NMEs and therapeutic biologics, to support these claims. Here, nanomedicines approved by the United States Food and Drug Administration's Center for Drug Evaluation and Research (CDER) from 2011 to 2023 are systematically identified and categorized. Nanomedicines were identified as formulations where submicron particle size contributes to product function. Like biologics and NMEs, nanomedicines are most frequently approved for oncological indications. Notably, all anti‐cancer nanomedicines are indicated to treat orphan diseases, which is representative of the potential for nanomedicines to occupy pharmaceutical niches in treating diseases affecting smaller patient cohorts. The median estimated cost of pivotal trials for nanomedicines is 47% that of biologics and NMEs approved in the same timeframe. The findings indicate higher manufacturing costs in nanomedicine development may be mitigated by savings elsewhere, increasing translational viability.

Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Imaged capillary isoelectric focusing method development for charge variants of high DAR ADCs

BackgroundCharge heterogeneity is a critical quality attribute for therapeutic biologics including antibody-drug conjugates (ADCs). Developing an ion exchange chromatography (IEX) or an imaged capillary isoelectric focusing (icIEF) method for ADCs with high drug-to-antibody ratio (DAR) is challenging because of the increased hydrophobicity from the payload-linker, DAR heterogeneity, and payload-linker instability. A sub-optimal method can be poorly stability-indicating due to the inability to discern contributions from charge and size variants conjugated with different number of drugs/payloads. Systematic strategy and guidance on charge variant method development is highly desired for high DAR ADCs with various complex structures. ResultsThis work encompasses the development and optimization of icIEF methods for high DAR ADCs of various DAR values (4–8) and payload linker chemistry. Method optimization focuses on improving resolution and stability indicating capabilities and differentiating contributions from the protein and payload-linker. Types, proportion, and combination of solubilizers and carrier ampholytes, as well as focusing parameters were interrogated. Our findings show that the structural units of the linker, the DAR, and the payload chemistry prescribe the selection of buffer, solubilizer, and ampholyte. We demonstrate that a stronger denaturant or solubilizer is needed for high DAR ADCs with polyethylene glycol (PEG)-containing linker structure compared to peptide linker. For unstable payload-linker, buffer system enhances sample stability which is vital to method robustness. In addition, a longer isoelectric focusing time is necessary for an ADC than its corresponding antibody to reach optimal focusing. SignificanceTo the best of our knowledge, this is the first comprehensive study on icIEF method development for charge variant determination of high DAR ADCs with unique physicochemical properties.

Intestinal Lymphatic Biology, Drug Delivery and Therapeutics: Current status and Future directions.

Historically, the intestinal lymphatics were considered passive conduits for fluids, immune cells, dietary lipids, lipid soluble vitamins and lipophilic drugs. Studies of intestinal lymphatic drug delivery in the late 20th century focussed primarily on the drug physicochemical properties, especially high lipophilicity, that resulted in intestinal lymphatic transport. More recent discoveries have changed our traditional view by demonstrating that the lymphatics are active, plastic and tissue-specific players in a range of biological and pathological processes, including within the intestine. These findings have, in turn, inspired exploration of lymph-specific therapies for a range of diseases, as well as the development of more sophisticated strategies to actively deliver drugs or vaccines to the intestinal lymph, including a range of nanotechnologies, lipid prodrugs and lipid-conjugated materials that 'hitchhike' on lymphatic transport pathways. With the increasing development of novel biological therapeutics there has been interest in whether these novel therapeutics are absorbed and transported through intestinal lymph after oral administration. Here we review the current state of understanding of the anatomy and physiology of the gastrointestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. We summarise the current state-of-the-art approaches to deliver drugs and quantify their uptake into the intestinal lymphatic system. Finally, and excitingly, we discuss recent examples of significant pharmacokinetic and therapeutic benefits achieved via intestinal lymphatic drug delivery. We also propose approaches to advance the development and clinical application of intestinal lymphatic delivery strategies into the future. Significance Statement This comprehensive review details understanding of the anatomy and physiology of the intestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. It highlights current state-of-the-art approaches to deliver drugs to the intestinal lymphatics and the shift toward the use of these strategies to achieve pharmacokinetic and therapeutic benefits for patients.

Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1.

Combinatorial antiretroviral therapy (cART) has transformed HIV infection from a death sentence to a controllable chronic disease, but cannot eliminate the virus. Latent HIV-1 reservoirs are the major obstacles to cure HIV-1 infection. Previously, we engineered exosomal Tat (Exo-Tat) to reactivate latent HIV-1 from the reservoir of resting CD4+ T cells. Here, we present an HIV-1 eradication platform, which uses our previously described Exo-Tat to activate latent virus from resting CD4+ T cells guided by the specific binding domain of CD4 in interleukin 16 (IL16), attached to the N-terminus of exosome surface protein lysosome-associated membrane protein 2 variant B (Lamp2B). Cells with HIV-1 surface protein gp120 expressed on the cell membranes are then targeted for immune cytolysis by a BiTE molecule CD4-αCD3, which colocalizes the gp120 surface protein of HIV-1 and the CD3 of cytotoxic T lymphocytes. Using primary blood cells obtained from antiretroviral treated individuals, we find that this combined approach led to a significant reduction in replication-competent HIV-1 in infected CD4+ T cells in a clonal in vitro cell system. Furthermore, adeno-associated virus serotype DJ (AAV-DJ) was used to deliver Exo-Tat, IL16lamp2b and CD4-αCD3 genes by constructing them in one AAV-DJ vector (the plasmid was named pEliminator). The coculture of T cells from HIV-1 patients with Huh-7 cells infected with AAV-Eliminator viruses led to the clearance of HIV-1 reservoir cells in the in vitro experiment, which could have implications for reducing the viral reservoir in vivo, indicating that Eliminator AAV viruses have the potential to be developed into therapeutic biologics to cure HIV-1 infection.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening

BackgroundPersonalized disease models are crucial for evaluating how diseased cells respond to treatments, especially in case of innovative biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells (nKPCs).MethodsEVs were isolated from nKPCs derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients’ urine and a line of Alport syndrome patient podocytes were characterized and used to assess albumin permeability in response to nKPC-EVs or various drugs. RNA sequencing was conducted to identify commonly modulated pathways after nKPC-EV treatment. siRNA transfection was used to demonstrate the involvement of SUMO1 and SENP2 in the modulation of permeability.ResultsTreatment with the nKPC-EVs significantly reduced permeability across all the steroid-resistant patients-derived and Alport syndrome-derived podocytes. At variance, podocytes appeared unresponsive to standard pharmacological treatments, with the exception of one line, in alignment with the patient’s clinical response at 48 months. By RNA sequencing, only two genes were commonly upregulated in nKPC-EV-treated genetically altered podocytes: small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2). SUMO1 and SENP2 downregulation increased podocyte permeability confirming the role of the SUMOylation pathway.ConclusionsnKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocytes with genetic dysfunction, through modulation of SUMOylation, an important pathway for the stability of podocyte slit diaphragm proteins. Our findings also suggest the feasibility of developing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

AAV-aMTD-Parkin, a therapeutic gene delivery cargo, enhances motor and cognitive functions in Parkinson's and Alzheimer’s diseases

Neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD), have a global prevalence and profoundly impact both motor and cognitive functions. Although adeno-associated virus (AAV)-based gene therapy has shown promise, its application for treating central nervous system (CNS) diseases faces several challenges, including effective delivery of AAV vectors across the blood-brain barrier, determining optimal dosages, and achieving targeted distribution. To address these challenges, we have developed a fusion delivery therapeutic cargo called AAV-aMTD-Parkin, which combines a hydrophobic cell-penetrating peptide sequence with the DNA sequences of AAV and Parkin. By employing this fusion delivery platform at lower dosages compared to zolgensma, we have achieved significant enhancements in cell and tissue permeability, while reducing the occurrence of common pathological protein aggregates. Consequently, motor and cognitive functions were restored in animal models of PD and AD. With its dual functionality in addressing PD and AD, AAV-aMTD-Parkin holds immense potential as a novel class of therapeutic biologics for prevalent CNS diseases.